This research was made possible by the Aortic Research Grant (University of Michigan) to Dr. Eliason. The authors would also like to acknowledge Nick Scott at the University of Michigan Plant Operations Sign and Graphics Department for assisting with the design and assembly of the cigarette lighting device.

The following protocol has been performed under the guidance and approval of the University of Michigan Institutional Animal Care and Use Committee (IACUC).
1. Electronic Cigarette-vapor Chamber Assembly
NOTE: The complete chamber should be placed in a fume hood during use. The chamber here was housed in a temperature controlled and filtered laboratory environment. Investigators may elect to monitor such aspects of the system to ensure consistency of the room air quality. As an option, covering the monitors with a metal cage can&#160;prevent rodent tampering while allowing the monitors to sample the interior chamber environment.&#160;
<ol>
	<li>Acquire an anesthesia chamber with air tight removable lid with volume of 20 L.</li>
	<li>Using a jig saw fitted with a blade appropriate for cutting the material, cut a 10.2 cm diameter hole in the lid of the chamber approximately 7.6 cm from the back edge of the chamber.</li>
	<li>Insert the adjustable vent into the hole and mount into place with any caulk adhesive. 
	NOTE: Ensure that the adhesive caulk is not accessible to the rodents in the chamber, as they may chew the caulk. To prevent this possible issue, apply the caulk to the outside of the chamber wall to mount the vent.</li>
	<li>Cut silicone tubing into two 15 cm segments, and attach ends to either side of a T-connector. 
	NOTE: Silicone tubing has the potential to react with some components of e-vapor or cigarette smoke. Thus, investigators may consider using non-reactive tubing.</li>
	<li>Thread both of the silicone tubes through premade holes near the front of the chamber lid so that the T-connector is inside the chamber. Ensure that the tubing is secured to the lid with either adhesive caulk or electrical tape.</li>
	<li>Connect the free ends of the silicone tubing to the output ends of two micro air pumps. Pumps should be mounted to the lid of the chamber with double-sided adhesive tape or caulk. 
	NOTE: The length of tubing connected to the output end of the pump should be short to limit the amount of vapor collection on the inside surface of the tubing during use.</li>
	<li>With a new silicone tube, attach one end to the input side of one of the air pumps (Pump A in Figure 1), and cut this tube to approximately 4 cm in length. This is where the electronic cigarette will be inserted during chamber use. Ensure that the diameter of the tube allows for a snug fit around the end of the e-cig.</li>
	<li>With a new silicone tube, attach one end to the input side of the other air pump (Pump B in Figure 1). This pump will introduce room air into the chamber. As such, the end of the tube must be placed outside the fume hood. The length of this tube is not critical, but should be as short as possible to limit airflow resistance.</li>
	<li>Inside the chamber attach two small hooks with double-sided adhesive to hold oxygen and carbon monoxide gas monitors.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/55672/55672fig1.jpg" /> 
Figure 1. Schematic of Electronic Cigarette-vapor Chamber. 
Chamber is housed in fume hood (not shown). Room air pump (Pump B) introduces room air from outside the fume hood into chamber continuously at 2 L/min. E-cig pump (Pump A) puffs 133 mL of E-vapor over 4 s, with a 30 s rest interval. E-vapor and room air mix prior to being pumped into chamber. Gas monitors continuously measure carbon monoxide (CO) and oxygen (O2) concentrations inside the chamber. E-vapor is exhausted passively through vent into fume hood. <a href="https://www.jove.com/files/ftp_upload/55672/55672fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Cigarette Smoke-chamber Assembly
NOTE: Virtually any brand of cigarette can be used with this system, however standardized research cigarettes such as the University of Kentucky 1R6F Research Cigarette are cost-effective, reliable, and best for this application.
<ol>
	<li>Follow steps 1.1 - 1.6, &#38; step 1.9.</li>
	<li>With a new silicone tube, attach one end to the cigarette lighting device, and the other end to the input side of the air pump (Pump A in Figure 2). The cigarette lighting device should be placed inside the fume hood and outside the chamber during use. 
	Note: Construction of the cigarette lighting device requires knowledge of metal fabrication and electrical engineering. While a step-by-step guide to construction will not be given here, see supplementary materials for plans.</li>
	<li>With a new silicone tube, attach one end to the other air pump (Pump B in Figure 2). This pump will introduce room air into the chamber, thus the end of the tube must be placed outside the fume hood.</li>
	<li>Cut several 5 mm wide vertical slits&#160;in the front wall of the chamber, and mount a computer fan on the outside of the chamber so that it covers this opening. Ensure that the front of the fan is facing towards the chamber such that the fan will blow air into the chamber through this opening.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/55672/55672fig2.jpg" /> 
Figure 2. Schematic of Cigarette-smoke Chamber. 
Room air pump (Pump B) introduces room air from outside the fume hood into chamber continuously at 2 L/min. Pump A draws on lit cigarette for 40 s with rate of 2 L/min, and 20 s later the computer fan evacuates the chamber over 3 min. Smoke and room air mix prior to being pumped into chamber. Gas monitors continuously measure carbon monoxide (CO) and oxygen (O2) chamber concentrations. Smoke is exhausted through vent into fume hood. <a href="https://www.jove.com/files/ftp_upload/55672/55672fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Microcontroller Assembly and Software
<ol>
	<li>Control the pumping system of the E-cigarette&#160;vapor chamber and cigarette smoke chamber by separate microcontrollers. Download the microcontroller software and upload the operating codes provided in the <a href="https://www.jove.com/files/ftp_upload/55672/supplementary_materials.zip" target="_blank">Supplementary Materials</a>. The electronic cigarette vapor code will run the room air pump continuously and activate the E-cigarette&#160;pump for a 4 s duration every 30 s. The cigarette smoke code will run the room air pump continuously, activate the cigarette pump for 40 s, and activate the computer fan 20 s after the cigarette pump has stopped. The fan will shut off after running for 3 min. 
	Note: The timing of the pumps and fan can be adjusted as needed. Reference manufacturer instructions on how to upload relevant codes to the microcontroller.</li>
	<li>To assemble the microcontroller, connect zip wires, diodes, resisters, and capacitors to the bread board as depicted in Figure 3 and attach alligator-clip wires to the corresponding air pumps (and computer fan for cigarette smoke chamber). House the microcontroller outside of the fume hood if possible.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/55672/55672fig3.jpg" /> 
Figure 3. Schematic of Microcontroller. 
Schematic of microcontroller and bread board to operate timing of air pumps and fan. <a href="https://www.jove.com/files/ftp_upload/55672/55672fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Animals
<ol>
	<li>Use adult rats weighing 450 - 520 g.</li>
	<li>Group rats according to exposure type (e.g., electronic cigarette, cigarette smoke, room air pumped into chamber).</li>
	<li>At this time prior to exposure, collect 500 &#181;L of blood in EDTA coated tubes from tail vein using syringe to measure baseline serum cotinine concentration.</li>
	<li>Spin down blood samples at 20,000 x g for 30 min at 4 &#730;C and collect serum. Ensure that samples are chilled on ice throughout this process.</li>
	<li>Following the manufacturer protocol, assay for cotinine in the serum samples collected. Samples may also be stored at -80 &#730;C for later use.</li>
</ol>
5. Operating the Electronic cigarette Chamber
<ol>
	<li>Clean the inside of the chamber with 70% ethanol, then with deionized water, and let air dry until the chamber is completely dry (or about 30 min).</li>
	<li>Calibrate gas monitors and mount entire monitor on the wall inside the chamber.</li>
	<li>Place rats inside the chamber. Note that up to three animals can be exposed simultaneously.</li>
	<li>Ensure the E-cigarette&#160;is fully charged with adequate E-liquid and insert the E-cigarette&#160; into input tube. It is critical to ensure that the E-cigarette&#160;battery and E-liquid levels are adequate throughout the duration of the 90-min exposure.</li>
	<li>Turn the air pumps on, and start the timer.</li>
	<li>During exposure observe the gas monitors to ensure chamber contains &#62;20% O2 and 0 ppm CO. 
	Note: If oxygen levels fall, the chamber may not be well ventilated or the oxygen monitor may not be appropriately calibrated.</li>
	<li>Once the exposure time has reached 90 min, remove the E-cigarette&#160;and continue to run the gas pumps to vent the remaining vapor. Additionally, the top of the chamber can be lifted to hasten ventilation.</li>
	<li>Once the vapor has cleared, remove the rats and clean the chamber.</li>
	<li>Collect 500 &#181;L blood from tail vein from each rat approximately 1 h after exposure at the conclusion of the experimental protocol.</li>
	<li>Follow steps 4.4 -&#160;4.5 to isolate serum and assay for cotinine.</li>
</ol>
6. Operating the Tobacco Cigarette Chamber
<ol>
	<li>Follow steps 5.1 - 5.3.</li>
	<li>Insert cigarette into cigarette lighting device, with the end of the cigarette against the heating elements.</li>
	<li>Turn on the cigarette lighting device until cigarette butt begins to smolder (about 5 s).</li>
	<li>Once the cigarette is lit, turn on the pumping system, start the timer, and observe the cigarette burn to completion (about 40 s).</li>
	<li>Once the cigarette has burned, carefully remove the spent cigarette from the cigarette lighting device with forceps.</li>
	<li>Ensure that CO levels do not rise above 1000 ppm and O2 levels do not fall below 20%. Computer fan timing and duration is critical in preventing carbon monoxide accumulation.</li>
	<li>After 4 min turn the pumping system off and return to step 6.2 until rats are exposed to tobacco smoke for 90 min (or approximately 23 cigarettes). 
	NOTE: Carbon monoxide levels 4 min following cigarette burning should fall below 400 ppm, otherwise carbon monoxide may begin to accumulate in chamber.</li>
	<li>When exposure is complete, continue to leave the pumping system on to ventilate residual smoke. When carbon monoxide falls below 100 ppm remove rats from chamber. This should take 5 - 10 min.</li>
	<li>Remove rats and clean the chamber.</li>
	<li>Collect 500 &#181;L blood from tail vein approximately 1 h after exposure at the conclusion of the experimental protocol as with the E-cigarettes.</li>
	<li>Follow steps 4.4 - 4.5 to isolate serum and assay for cotinine.</li>
</ol>

<ol>
	<li>Schoenborn, C. A., Gindi, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electronic+Cigarette+Use+Among+Adults:+United+States,+2014+Key+findings.">Electronic Cigarette Use Among Adults: United States, 2014 Key findings.</a> NCHS. , (2014).</li><li>Singh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+Use+Among+Middle+and+High+School+Students+-+United+States,+2011-2015.">Tobacco Use Among Middle and High School Students - United States, 2011-2015.</a> MMWR. 65 (14), 361-367 (2016).</li><li>Flora, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+potential+impurities+and+degradation+products+in+electronic+cigarette+formulations+and+aerosols.">Characterization of potential impurities and degradation products in electronic cigarette formulations and aerosols.</a> Regul. Toxicol. Pharmacol: RTP. 74, 1-11 (2016).</li><li>Tierney, P. A., Karpinski, C. D., Brown, J. E., Luo, W., Pankow, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavour+chemicals+in+electronic+cigarette+fluids.">Flavour chemicals in electronic cigarette fluids.</a> Tob. Control. , (2015).</li><li>Sleiman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emissions+from+Electronic+Cigarettes:+Key+Parameters+Affecting+the+Release+of+Harmful+Chemicals.">Emissions from Electronic Cigarettes: Key Parameters Affecting the Release of Harmful Chemicals.</a> Environ. Sci. Technol. 50 (17), 9644-9651 (2016).</li><li>Hwang, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electronic+cigarette+inhalation+alters+innate+immunity+and+airway+cytokines+while+increasing+the+virulence+of+colonizing+bacteria.">Electronic cigarette inhalation alters innate immunity and airway cytokines while increasing the virulence of colonizing bacteria.</a> Int J Mol Med (Berlin, Germany). 94 (6), 667-679 (2016).</li><li>Cheng, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+evaluation+of+electronic+cigarettes.">Chemical evaluation of electronic cigarettes.</a> Tob Control. 23, 11-18 (2014).</li><li>Etter, J. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+longitudinal+study+of+cotinine+in+long-term+daily+users+of+e-cigarettes.">A longitudinal study of cotinine in long-term daily users of e-cigarettes.</a> Drug Alcohol Depend. 160, 218-221 (2016).</li><li>Etter, J. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Levels+of+saliva+cotinine+in+electronic+cigarette+users.">Levels of saliva cotinine in electronic cigarette users.</a> Addiction. 109 (5), 825-829 (2014).</li><li>Marsot, A., Simon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+and+Cotinine+Levels+With+Electronic+Cigarette:+A+Review.">Nicotine and Cotinine Levels With Electronic Cigarette: A Review.</a> Int. J. Toxicol. 35 (2), 179-185 (2016).</li><li>Flouris, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+impact+of+active+and+passive+electronic+cigarette+smoking+on+serum+cotinine+and+lung+function.">Acute impact of active and passive electronic cigarette smoking on serum cotinine and lung function.</a> Inhal. Toxicol. 25 (2), 91-101 (2013).</li><li>Bot, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+cotinine+levels+in+cigarette+smokers:+impact+of+mental+health+and+other+correlates.">Plasma cotinine levels in cigarette smokers: impact of mental health and other correlates.</a> Eur Addict Res. 20 (4), 183-191 (2014).</li><li>Zhu, S. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four+hundred+and+sixty+brands+of+e-cigarettes+and+counting:+implications+for+product+regulation.">Four hundred and sixty brands of e-cigarettes and counting: implications for product regulation.</a> Tob. Control. 23, 3-9 (2014).</li></ol>

The authors have nothing to disclose.

Here we describe a method for constructing chambers that expose rodents to E-vapor and cigarette smoke in a controlled fashion (Figure 6). Construction of the E-cigarette chamber is relatively simple and inexpensive compared to commercial exposure systems14,15,16. The parts and tools required to build the chamber are readily available from commercial suppliers online. Similarly, constructing the cigarette smoke ...

Since entering the US market in 2004, electronic cigarettes (E-cigarettes) have expanded into a billion-dollar industry, and it is estimated that nearly 9 million adults use them regularly1. In 2014 and 2015, more high school students had used E-cigarettes than conventional cigarettes2. The growing number of E-cigarettes users has spawned a research effort to evaluate their potential adverse health effects.
E-cigarettes generate a vapor (dubbed &#34;E-vapor&#34;) by heating a viscous solution that typically contains a mixture of water, polyethylene glycol or vegetable glycerin, nicotine, and flavorings3,4. It has been shown that E-vapor contains several harmful compounds including Reactive Oxygen Species (ROS), nicotine, various aldehydes, and polycyclic aromatic hydrocarbons5,6. Many of these compounds are formed during the vaporization process of E-liquid prior to inhalation7. Notably, several of these harmful compounds are also present in cigarette smoke, raising concern that E-cigarettes use may have similar adverse health consequences7.
There is little consensus on the health effects of E-cigarettes. To address this, several animal models of E-vapor exposure have been developed (Table 1). These models employ a variety of methods such as whole-body E-vapor exposure and mechanical ventilation. While current models have provided insightful data, few make direct comparisons to cigarette smoke within the same exposure system (Table 1). Additionally, while several human studies have shown E-cigarettes&#160;users and cigarette smokers to have serum cotinine levels between 30-200 ng/mL, many models of E-vapor and cigarette smoke exposure fall outside this range8,9,10,11,12.
Herein we present a method for comparing the effects of cigarette smoke and E-vapor exposure in vivo that yields serum cotinine levels similar to human studies.

<table border="0" cellpadding="0" cellspacing="0" width="793">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:348px;">blu PLUS Rechargeable Kit</td>
			<td style="width:219px;">blu eCigs</td>
			<td style="width:119px;">N/A</td>
			<td style="width:107px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1R6F Reference Cigarettes</td>
			<td>Center for Tob Ref&#160; Prod UK</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lexan Anesthesia Chamber 20 L</td>
			<td>Jorgensen Laboratories</td>
			<td>JOR265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Arduino UNO</td>
			<td>Arduino</td>
			<td>2877</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diode Rectifier - 1A 50V</td>
			<td>Spark Fun</td>
			<td>COM-08589</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Resistor 10K Ohm 1/6th Watt PTH - 20 pack</td>
			<td>Spark Fun</td>
			<td>COM-11508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electrolytic Decoupling Capacitors - 100uF/25V</td>
			<td>Spark Fun</td>
			<td>COM-00096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solderless Plug-in BreadBoard&#160;</td>
			<td>BusBoard Prototype Systems</td>
			<td>BB400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alligator-Clip Wires</td>
			<td>BusBoard Prototype Systems</td>
			<td>CA-M-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ZipWire</td>
			<td>BusBoard Prototype Systems</td>
			<td>ZW-MM-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard Fan 80 ST2</td>
			<td>Cooler Master</td>
			<td>R4-S8R-20AK-GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ARIC 4&#34; adjustable vent</td>
			<td>Bestlouver</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ToxiPro&#160; Carbon Monoxide Monitor</td>
			<td>Honeywell Analytics</td>
			<td>54-00-10316</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ToxiPro Oxygen Monitor</td>
			<td>Honeywell Analytics</td>
			<td>54-45-90-VD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ToxiPro IQ Express Docking Station</td>
			<td>Honeywell/Sperian Biosystems&#160;</td>
			<td>54-46-9100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Command Wall Hook Small Wire 6-Pack</td>
			<td>3M</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Water/Air Pump</td>
			<td>Xiamen Conjoin Electronics</td>
			<td>CJWP40-A12A1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1/4&#34; Silicon Tubing</td>
			<td>NewAge</td>
			<td>2801470-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T Connector</td>
			<td>Bel-Art Scienceware</td>
			<td>F196060000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic Whole Blood tube with spray-coated K2EDTA</td>
			<td>Becton, Dickinson and Company</td>
			<td>367841</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cotinine ELISA kit</td>
			<td>Calbiotech</td>
			<td>CO096D</td>
			<td></td>
		</tr>
	</tbody>
</table>

comparing the effects of electronic cigarette vapor and cigarette smoke in a novel in vivo exposure system

Electronic cigarettes (E-cigarettes) are being widely used, and growing in popularity. It is estimated that more than 9 million adults use them regularly. The potential adverse health effects of electronic cigarette vapor (E-vapor) exposure are poorly defined. While several animal models of E-vapor exposure have been developed, few models expose rodents to clinically relevant quantities of nicotine and make direct comparisons to cigarette smoke within the same exposure system. Here, we present a method for constructing and operating an E-vapor chamber and cigarette smoke chamber. The chambers are constructed by outfitting anesthesia chambers with a computer controlled pumping system that delivers consistent amounts of E-vapor or cigarette smoke to rodents. Nicotine exposure is measured indirectly by quantifying pre and post-exposure serum cotinine levels. This exposure system can be modified to accommodate various types of E-cigarettes&#160;and tobacco cigarettes, and can be used to compare the effects of E-vapor&#160;and cigarette smoke in vivo.

Carbon monoxide and oxygen monitoring
Oxygen concentrations did not fall below 20% during e-vapor exposure and CO concentrations remained undetectable throughout exposure. Gas monitors during cigarette smoke exposure indicated that oxygen concentration remained above 20%. Carbon monoxide concentrations did not exceed 1,000 ppm (Figure 4).
<img alt="Figure...

Watch this Scientific Journal Video about Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System at JoVE.com

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

This protocol describes a method for exposing rodents to electronic cigarette vapor (E-vapor) and cigarette smoke. Exposure chambers are constructed by modifying anesthesia chambers with an automated pumping system that delivers E-vapor or cigarette smoke to rodents. This system can easily be modified to accommodate many experimental endpoints.

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

Electronic cigarettes (E-cigarettes) are being widely used, and growing in popularity. It is estimated that more than 9 million adults use them regularly. ...

comparing-effects-electronic-cigarette-vapor-cigarette-smoke-novel

Research

JoVE Journal

Medicine

11.4K Views.  University of Michigan Medical School. This protocol describes a method for exposing rodents to electronic cigarette vapor (E-vapor) and cigarette smoke. Exposure chambers are constructed by modifying anesthesia chambers with an automated pumping system that delivers E-vapor or cigarette smoke to rodents. This system can easily be modified to accommodate many experimental endpoints.

Video: Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated respiratory epithelium. A protocol is described for isolating and exposing these cells to mainstream cigarette smoke (CS), in order to study epithelial cell responses to CS exposure. The protocol consists of three parts: the isolation of airway epithelial cells from mouse trachea, the culturing of these cells at air-liquid interface (ALI) as fully differentiated epithelial cells, and the delivery of calibrated mainstream CS to these cells in culture. The ALI culture system allows the culture of respiratory epithelia under conditions that more closely resemble their physiological setting than ordinary liquid culture systems. The study of molecular and lung cellular responses to CS exposure is a critical component of understanding the impact of environmental air pollution on human health. Research findings in this area may ultimately contribute towards understanding the etiology of chronic obstructive pulmonary disease (COPD), and other tobacco-related diseases, which represent major global health problems.

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated ...

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...